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Not applicable.
The present invention is in the field of molecular biology. More specifically, the invention is in the field of assays that utilize fluorescently-labeled probes and primers in hybridization assays for detection of nucleic acids.
The use of fluorescent molecules in the biological sciences for research and diagnostic purposes is well known. See, for example, Kirkbright xe2x80x9cFluorescent Indicatorsxe2x80x9d in Indicators, (ed. Bishop, E.) Pergamon Press, New York, Chapter 9, pp. 685-708, 1972; and Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals, Sixth edition, Molecular Probes, Inc., Eugene, Oreg. Fluorescent moieties have been used for non-specific labeling of single- and double-stranded nucleic acids (e.g., acridine, ethidium bromide) and for labeling of nucleic acid probes that are used in sequence-specific detection of nucleic acid targets. In general, when fluorescent nucleic acid binding molecules and/or fluorescently-labeled probes are used for nucleic acid detection, unbound fluorescent material must be removed from the system, prior to analysis, to maximize detection of a signal. If unbound material is not removed, background fluorescence leads to a reduction in the signal:noise ratio.
Compositions which are fluorescent when bound to double-stranded DNA, but which do not fluoresce (or fluoresce at a different wavelength) when unbound, have been described. See, for example, Haugland, supra, pp. 144-156 and 161-174, especially pp. 161-165. Although such compositions may exhibit fairly general sequence preferences (e.g., for AT-rich vs. GC-rich target sequences), they are not capable of either sequence-specific detection of a target or of mismatch discrimination between targets having related but non-identical sequences. In addition, such compositions cannot be used for multiplex detection of target sequences (i.e., simultaneous detection of more than one target sequence).
Several new analytical techniques depend on sequence-specific detection and mismatch discrimination using fluorescence as a readout. For instance, homogeneous detection methods for monitoring the accumulation of specific PCR products have recently been developed. One of these assays utilizes an oligonucleotide probe which contains a fluorescent molecule at its 5xe2x80x2 end and a fluorescence quencher at its 3xe2x80x2 end. Because of the presence of the quencher, the oligonucleotide probe does not exhibit fluorescence, or exhibits relatively low fluorescence, in the single-stranded state. The assay exploits the 5xe2x80x2xe2x86x923xe2x80x2 nuclease activity of Taq DNA polymerase to hydrolyze such a probe after it has formed a sequence-specific duplex with a target nucleic acid. Hydrolysis releases the fluorescent molecule from the 5xe2x80x2 end of the probe, removing it from proximity with the quencher, thereby allowing increased fluorescence to occur. Lee et al. (1993) Nucleic Acids Res. 16:3761-3766. In another recently-developed technique, microvolume multi-sample fluorimeters with rapid temperature control have been developed for use with 5xe2x80x2-nuclease assays using double-labeled fluorescent probes. Wittwer et al. (1997) Biotechniques 22:176-181. U.S. Pat. No. 5,871,908 describes a homogeneous assay in which fluorescent signal varies with a temperature gradient and the variation is detected in real time. However, all of these assays involve post-hybridization detection steps, often involving the use of enzymes, which are costly, time-consuming and can be difficult to regulate, in terms of their activity.
There is thus a need for sensitive and straightforward methods and compositions for sequence-specific detection of nucleic acid targets; in particular fluorescent detection. Besides the advantages of using fluorescent molecules as an alternative to radioisotopes, improvements in speed, economy and convenience would attend the development of a method in which the hybridization event itself provided a direct readout, without requiring subsequent detection steps, such as enzymatic treatment of hybridized material.
Tyagi et al. (1996) Nature Biotechnol 14:303-308 described probes containing a fluorophore and a quencher molecule which, in the unhybridized state, form a hairpin which brings the fluorophore and the quencher into proximity so that fluorescence is quenched. Upon hybridization, the hairpin structure is disrupted and fluorescence is observed. Such probes require the attachment of both a fluorophore and a quencher, and also must contain regions of self-complementarity, which may interfere with their ability to hybridize to their target.
Minor groove binding agents that non-covalently bind within the minor groove of double stranded DNA have been described. Zimmer et al. (1986) Prog. Biophys. Molec. Biol. 47:31-112; Levina et al. (1996) Antisense and NucL. Acid Drug Develop. 6:75-85. Hybridization assays using an oligonucleotide coupled to a minor groove binder (MGB) have been described in U.S. Pat. No. 5,801,155, and in International Patent Application No. PCT/US99/07487. These publications describe the ability of minor groove binders, when conjugated to an oligonucleotide, to increase the ability of the oligonucleotide to distinguish between a perfectly-matched target sequence and a target sequence with a single-nucleotide mismatch. This heightened discriminatory ability of MGB-oligonucleotide conjugates is reflected in a greater difference in melting temperature (Tm) between matched and mismatched duplexes formed with an MGB-oligonucleotide conjugate, on the one hand, and matched and mismatched duplexes formed with an unmodified oligonucleotide, on the other. The aforementioned U.S. Pat. No. 5,801,155, and International Patent Application No. PCT/US99/07487 additionally disclose that a duplex comprising a MGB-oligonucleotide conjugate has a higher melting temperature than a duplex of identical sequence comprising an unmodified oligonucleotide. This property of duplexes comprising a MGB-oligonucleotide conjugate allows more facile detection of related mismatched sequences with a MGB-oligonucleotide probe, and enables the use of shorter oligonucleotide probes in PCR amplification reactions, if the probe is conjugated to a MGB. These publications also describe the use of an oligonucleotide coupled to a minor groove binder, a fluorophore and a fluorescent quencher, in hydrolyzable probe assays.
Intercalating agents are, generally speaking, flat aromatic molecules that bind non-covalently to double-stranded DNA or RNA by positioning themselves between adjacent base pairs of the duplex. Gago (1998) Method 14:277-292. U.S. Pat. No. 4,835,283 and PCT publication WO 98/50541, for example, disclose oligonucleotides that are covalently bound to an intercalating group. Oligonucleotides conjugated to either minor groove binders or intercalating groups can be used in hybridization assays.
Hoechst 33258 and 33342 are examples of fluorescent dyes that bind in the minor groove of DNA duplexes. A conjugate consisting of an oligonucleotide coupled to a Hoechst-like minor groove binder has been observed to show increased fluorescence upon hybridization to a single-stranded target. O""Donnell et al. (1995) Biorg. Med. Chem. 3:743-750; and Wiederholt et al. (1996) J. Amer. Chem. Soc. 118:7055-7062. This conjugate consisted solely of an oligonucleotide bound to a MGB.
EP 231 495 discloses a polynucleotide compound comprising at least two entities, which upon hybridization is capable of generating a change in property of the hybrid.
The present invention provides methods and compositions for improved hybridization detection and mismatch discrimination by fluorescence. In the practice of the invention, an increase in fluorescent signal, a change in fluorescence excitation and/or emission, and/or some other change in fluorescence properties occurs after hybridization of an oligonucleotide, appropriately labeled with a latent fluorophore and a minor groove binder, to a complementary target.
In one aspect, the present invention encompasses a covalently bound oligonucleotide (ODN)/minor groove binder (MGB)/latent fluorophore (LF) combination. The oligonucleotide comprises a plurality of nucleotides (and/or modified nucleotides and/or nucleotide analogues), a 3xe2x80x2 end and a 5xe2x80x2 end. A minor groove binder moiety is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which molecule binds in a non-intercalating manner into the minor groove of non-single-stranded nucleic acids or hybrids, analogues and chimeras thereof (i.e., double- or triple-stranded polynucleotides) with an association constant greater than approximately 103Mxe2x88x921. The minor groove binder moiety is covalently attached at the 3xe2x80x2 end and/or the 5xe2x80x2 end, and/or to at least one of said nucleotides, modified nucleotides and/or nucleotide analogues of the oligonucleotide, and is typically attached to the oligonucleotide through a first linking group having a backbone length of no more than about 100 atoms. A latent fluorophore is a radical of a molecule having a molecular weight of approximately 150 to approximately 5000 Daltons which binds in an intercalating manner into non-single-stranded nucleic acids or hybrids, analogues and chimeras thereof, or lies preferentially in the minor groove, or in another manner is oriented to the DNA molecule by the minor groove binder moiety so that it becomes fluorescent or its fluorescence properties are changed in a detectable way. Typically, the latent fluorophore is attached to the minor groove binder moiety through a second linking group having a backbone length of no more than about 50 atoms.
In one embodiment, the ODN-MGB-LF conjugate is relatively non-fluorescent in its single-stranded state, but becomes fluorescent after hybridization to a target sequence. In another embodiment, the ODN-MGB-LF conjugate may exhibit some fluorescence emission at one or more particular wavelengths in its single-stranded state, but, after hybridization, its maximal fluorescence emission is shifted to a different wavelength. In yet another embodiment, the wavelength at which maximal fluorescence excitation occurs can change after hybridization of an ODN-MGB-LF conjugate.
In another aspect, the present invention encompasses processes for the synthesis of covalently-bound oligonucleotide-minor groove binder-latent fluorophore conjugates. The invention also provides novel compositions for use in the synthesis of ODN-MGB-LF conjugates.
In yet another aspect, the invention relates to the use of compositions comprising an oligonucleotide, a minor groove binder and a latent fluorophore, in covalent or functional linkage, as hybridization probes for fluorescent detection in analytical and diagnostic methods. These methods include but are not limited to, PCR (including real-time PCR), single nucleotide mismatch discrimination, target amplification, signal amplification and assays utilizing oligonucleotide arrays.
In an exemplary method for detecting a target sequence in a polynucleotide, an ODN-MGB-LF conjugate is combined with a sample containing a polynucleotide to form a hybridization mixture, wherein the ODN portion of the conjugate comprises a sequence which hybridizes to the target sequence, the hybridization mixture is incubated under conditions which yield specific hybridization, and thereafter fluorescence of the hybridization mixture is measured, wherein fluorescence is indicative of the presence of the target sequence.
In another embodiment, the compositions and methods of the invention are used for detection of a target sequence in a polynucleotide, wherein the polynucleotide is in a sample comprising a plurality of polynucleotides having different sequences.
In yet another embodiment, the compositions and methods of the invention are used for detection of a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence. In this embodiment, an ODN-MGB-LF conjugate is contacted with the aforementioned mixture of polynucleotides, wherein the ODN-MGB-LF forms a stable hybrid only with a target sequence that is perfectly complementary to the oligonucleotide portion of the composition and wherein the composition does not form a stable hybrid with any of the related sequences. After hybridization, the fluorescence of the mixture is measured, wherein fluorescence is indicative of the presence of the target sequence.
In a further embodiment, the compositions and methods of the invention are used for single-nucleotide mismatch discrimination.
In one embodiment, the compositions are used for the detection of single-stranded nucleic acids. The ODN portion of the ODN-MGB-LF conjugate forms a duplex with a single-stranded target nucleic acid, and interactions of the MGB and LF portions of the conjugate with the resulting duplex nucleic acid result in enhanced fluorescence, or some other change in the fluorescence properties of the latent fluorophore.
In another embodiment, the compositions of the invention are used for detection of double-stranded nucleic acid targets. In this case the ODN portion of the conjugate is a triplex-forming oligonucleotide. See, for example, Fresco, U.S. Pat. No. 5,422,251; Hogan, U.S. Pat. No. 5,176,996; and Lampe (1997) Nucleic Acids Res. 25:4123-4131. Formation of a triplex between the conjugate and a double-stranded target results in enhanced fluorescence, or some other change in the fluorescence properties of the latent fluorophore.
In another embodiment, the invention provides compositions and methods for the simultaneous detection of multiple target sequences in a sample (i.e., multiplex detection).
In another embodiment, the invention provides compositions and methods for amplification of a target sequence, wherein the amplification primer(s) are capable of hybridization-triggered fluorescence. This embodiment is particularly suitable for various amplification methods in which the product is detectable in real time.
In further aspects, ODN-MGB-LF conjugates are immobilized on a solid support, preferably in an ordered array. An immobilized conjugate can be used for capture of a target polynucleotide and/or as a primer using a captured polynucleotide as a template. In these and other applications, the compositions of the invention are able to discriminate between closely related polynucleotide sequences.
In another aspect, the invention provides kits for fluorescent detection of nucleic acids, and for mismatch discrimination between related nucleic acids, wherein the kits comprise at least one ODN-MGB-LF conjugate.